Concept Paper

MICROMORPHOLOGICAL COMPARISON ON AGOHO TREE (Casuarina equisetifolia) AND

PINE TREE (Pinus kesiya)
Fernandez, Patricia Barbara A., Flores, John Clarence H., Garcia, Josue S., Lopez, Julienne C.,
Medina, Renz Gil B.
Department of Biology, College of Science, University of the Philippines Baguio, Baguio City, 2600,
Benguet

I. Introduction

Benguet pine trees (Pinus kesiya) are one of the most abundant pines in the Philippines

(Luu & Thomas, 2004). They are described as conifers and gymnosperms that could reach a height

of up to 30 to 35 meters. Their barks are thick and leaves, needle-like. They have cones which

contains their reproductive structures (Farjon, 2013). Another tree species abundant in the country

as well are agoho trees (Casuarina equisetifolia). Agoho trees are members of the family,

Casuarinaceae. They are described as evergreen trees with a soft wispy pine-like appearance. They

bear resemblance to coniferous plants such as Pinus kesiya because of their production of cone-like

fruits and pine-needle-like leaves (Elfers, 1988) which people often mistake them for as conifers

as well.

With these morphological similarities, this paper aims to compare the micromorphological

differences between agoho trees (Casuarina equisetifolia) and pine trees (Pinus kesiya) in terms of

primary plant structures such as their leaves, stem, roots present in each specimen. It also aims to

qualitatively describe the different vegetative and reproductive parts of Casuarina equisetifolia and

Pinus kesiya both micro- and macroscopically, which could put further emphasis on their

differences.
 II. Materials and Methods

A. Materials

In the process of slide preparation, the following materials and solutions will be used:

Equipment: Solution:

● Aspirator ● Acetone

● Cork ● Cellulose acetate

● Slide ● 50 % Ethanol

● Coverslip ● 40% Ethanol

● Scalpel; No. 3 surgical knife handle with ● Formalin-acetic acid-alcohol

No. 10 blade ● 1% safranin

● Forceps ● 50% alcohol

Specimen: ● 95% alcohol

● Casuarina equisetifolia (stem, leaf, root) ● Haematoxylin

● Pinus kesiya (stem, leaf, root) ● Xylene

B. Air removal

To extract air from its cell lumina, the specimen would be soaked in water. For faster results, it is

advisable to alternate the cold and boiling temperature of water. Observe until the sinking of the lightest

block is achieved. Then, use the aspirator to ensure that little to no air comes from the tissues (Brown,

1919).

C. Softening of tissues

The specimens are to be submerged in pure acetone for 1 to 2 hours. Then, soak in a 12 percent

solution of cellulose acetate in acetone to soften. It would require 5 to 14 days depending on the hardwood

sections of the specimen. After softening, it should be placed in running water to remove the acid (McLean

& Cook, 1963).

 D. Slide preparation

A cross section for the stem and root is performed to accentuate the growth and development of the

specimen. For the wooden stem and root, it should be divided into small blocks, which are suitable to be

clamped by improvised microtome holders. Before using, the specimen should be submerged in water to

prevent further dehydration. Then, to fix the sample tightly, make a holder by using homogenous cork to

stabilize the specimen, specifically cutting it longitudinally into 2 halves. Then, make a groove inside that

can fit the specimen.

Use the surgical knife to make a cut perpendicular to the fibers. Specifically, make thin cut sections

from the end that is projected out by the microtome holder. For the leaf, a microtome holder is not necessary

due to its thinness and stability. The section can be acquired by providing a suitable support, specifically

holding down the leaf in place. Afterwards, sever a part of the leaf at the middle. Obtain several thin sections

to compare and contrast the optimum thickness for mounting. Use the forceps and fix the thin section using

formalin-acetic acid-alcohol solution.

Submerge the specimen into 40% ethanol for further dehydration. After 30 to 60 seconds, rinse the

specimen with 50% ethanol solution for another 30 to 60 seconds. Prepare a stain of 1% safranin in 50%

alcohol and haematoxylin. Place the solution in a petri dish, then transfer the section for 24 hours.

Transfer the sections in 50% alcohol. To prevent further decolorizing effect, place the sections in a

95% alcohol for about 5 minutes. For best results, choose sections that are darkly stained. Place the section

in a 95% alcohol for about a minute and transfer them in a 50:50 absolute alcohol and xylene mixture. Then

place the sections in xylene for 10 minutes.

Place the specimen on a slide. For determining the best for mounting, put a drop of xylene in the

specimen to avoid dehydration and view them under the microscope (Cutler, 2008); (Gärtner &

Schweingruber, 2013); (Zamora, 1992).

III. References

Brown, F. (1919). The Preparation and Treatment of Woods for Microscopic Study. Bulletin of the Torrey

Botanical Club, 46(4), 127-150. doi:10.2307/2479494

Cutler, D. F., et al. (2008). Plant Anatomy: An Applied Approach. Australia: Blackwell Publishing. p170-

194.

Elfers, S. (1988). Element stewardship abstract for Casuarina equisetifolia. Virginia, US: The Nature

Conservancy.

Farjon, A. (2013). Pinus kesiya. IUCN Red List of threatened species. Retrieved from:

https://dx.doi.org/10.2305/IUCN.UK.2013-1.RLTS.T42372A2975925.en

Gärtner, H. & Schweingruber, F. (2013). Microscopic Preparation Techniques for Plant Stem Analysis.

Switzerland: Swiss Federal Research Institute.

Luu, N. & Thomas, P. (2004). Conifers of Vietnam. England: UK Centre for Ecology & Hydrology.

McLean, R.C. & Cook, W.R. (1963). Plant science formulae: A reference book for plant science

laboratories (including Bacteriology). New York: St Martin's Press.

Zamora, C.V. (1992). Laboratory Manual in Plant Morpho-anatomy. Philippines: University of the

Philippines Press.